This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Azides, which are extremely rare in biological systems, are emerging as attractive chemical handles for bioconjugation. In particular, the Cu(I) catalyzed 1,3-dipolar cyclization of azides with terminal alkynes to give stable triazoles has been employed for tagging a variety of biomolecules including proteins, nucleic acids, lipids, and saccharides. The cycloaddition has also been used for activity-based protein profiling, monitoring of enzyme activity, and the chemical synthesis of microarrays and small molecule libraries. An attractive approach for installing azides into biomolecules is based on metabolic labeling whereby an azide-containing biosynthetic precursor is incorporated into biomolecules using the cells'biosynthetic machinery. This approach has been employed for tagging proteins, glycans, and lipids of living systems with a variety of reactive probes. These probes can facilitate the mapping of saccharide-selective glycoproteins and identify glycosylation sites. Alkyne probes have also been used for cell surface imaging of azide-modified biomolecules and a particularly attractive approach involves the generation of a fluorescent probe from a non-fluorescent precursor by a [3+2] cycloaddition. Despite many attractive features, a major disadvantage of the copper-catalyzed cycloaddition is the cellular toxicity of the metal catalyst, precluding applications wherein cells must remain viable. Hence, there is great need for the development of CuI free [3+2] cycloadditions. In this respect, alkynes can be activated by ring strain and, for example, constraining an alkyne within an eight membered ring creates 18 kcal/mol of strain, much of which is released in the transition state upon [3+2] cyclcoaddition with an azide. As a consequence, cyclooctynes react with azides at room temperature without the need of a catalyst. The strain-promoted cycloaddition has been used to label biomolecules without observable cyto-toxicitiy. The scope of the approach has, however, been limited due to the slow rate of reaction. Appending electron-withdrawing groups to the octyne ring can increase the rate of strain-promoted cycloadditions. However, this type of modification may make the alkyne prone to nucleophilic attack. Staudinger ligation with a phosphine reagent offers the most attractive reagent for cell surface labeling of azides.